This project will apply advanced and emerging technologies in proteomics to the discovery of novel molecular targets of blocking malaria transmission. While primarily exploratory, this proposal is also based on two hypotheses: 1) Plasmodium zygotes (the products of gamete fusion/fertilization in the mosquito midgut) express novel, perhaps low abundance, but hitherto unidentified proteins that may be immunological targets of blocking malaria transmission; and 2) Plasmodium ookinetes (the motile developmental stage that succeeds the zygote and actively invades the midgut) express novel secreted and cell surface-associated molecules that are specifically involved in parasite invasion of the mosquito midgut epithelium and are potential targets of blocking malaria transmission. There are two Specific Aims: 1) To delineate the proteome of Plasmodium gallinaceum zygotes and ookinetes using complementary techniques of multidimensional protein identification technology (MUDPIT), quantitative isotope-coded affinity tag mass spectrometry, and quantitative 2D gel electrophoresis, and use bioinformatics tools to comparatively identify zygote and ookinete stage genes in the extant Plasmodium genome databases, particular in the lethal human malaria parasite P. falciparum; and 2) To initiate the validation of zygote/ookinete-expressed proteins by testing a select number of newly identified molecules in an animal model of transmission-blocking immunity, both with a new DNA vaccine technology and traditional recombinant protein expression/vaccination. Data will be placed into the PlasmoDB public database. This application directly relates to the parent R01 grant AI45999, "Molecular Targets of Blocking Malaria Transmission," since the focus of the present application is on using proteomics to discover and validate new molecular targets of blocking malaria transmission. This proposal also fits well into the robust extant infrastructure supported by the parent R01 grant to test newly identified parasite molecules as targets of blocking malaria transmission. This application is consistent with the exploratory/development nature of the R21 mechanism since it proposes to apply new and emerging technologies to discovering unknown but potentially important and interesting Plasmodium molecules.